RNA-SeqTranscriptome Analysis of Spirodela Dormancy Without Reproduction and Identification of Molecular Targets Useful for Improving Biomass Production for Industrial Applications

ABSTRACT

Compositions and methods are provided for altering carbon partitioning in biomass isolated from Duckweed.

This application claims priority to U.S. Provisional Application No. 61/912,328 filed Dec. 5, 2013, the entire disclosure being incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the fields of plant molecular biology and recombinant manipulation of plant species in order to maximize production of biomass having desirable characteristics. More specifically, the invention provides valuable gene targets for manipulating carbon production in Duckweed based on results obtained from deep sequencing of the Duckweed genome.

BACKGROUND OF THE INVENTION

Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found within and at the end of the specification. The disclosure of each of these references is incorporated by reference.

Plants, unlike animals, do not have fur nor can they seek shelter to survive under food shortage and cold weather conditions. Consequently, they often become dormant to avoid adverse environments, such as poor nutrition, chilling temperature and drought. Dormancy is a complex state of plant development, in which the plant body exhibits little or no growth. Plants resume their growth once the conditions are favorable.

There are mainly two types of plant dormancy, e.g., forming seeds or buds. Seed dormancy has been observed for many plants species including our major crops [1-3]. Winter dormant buds are found for instance in woody plants, bulbs, rhizomes and tubers of herbaceous plants [4]. Studies on the molecular mechanisms of bud dormancy transitions in perennial woody plants have been conducted, including pear[5], oak[6], and poplar[7].

Spirodelapolyrhiza, a floating aquatic monocot, develops a specific dormant organ called a turion during its life cycle, which alternates between periods of clonal propagation and dormancy. Its leaf, stem and bud are extremely compact forming a round-shaped frond, resembling a single leaf. Large numbers of Spirodela plants can be maintained in cell cultures under totally controlled medium and environmental conditions. They reproduce vegetatively through budding of fronds (growth phase) during spring and summer[8] and transition to turions (dormant phase), when there is a shortage of nutrients in the fall or when the temperature drops in the winter[9].

Noticeably, fronds perform photosynthesis and turions function as storage for starch and germinate in the following spring[10-13]. Turion cells exhibit dense intercellular space, thick cell wall and are also rich in anthocyanins[14]. Therefore, turions provide a unique system to study both bud and seed dormancy because they reproduce like buds without sexual hybridization but are functionally equivalent to seeds that could generate a progeny plant in the growing season. Previous studies have shown that addition of abscisic acid (ABA) into growth medium quickly leads to turion formation after 5 days of treatment in the laboratory[13, 15, 16]. Only 3 days after ABA treatment, the Spirodelaprimordium is irreversibly committed to turion development[15]. The ease of growth and its direct contact with water make Spirodela a model system to gain molecular insights into plant dormancy[17].

At the molecular level, some studies on turion development have already been performed. For example, the transcript level of D-myo-inositol-3-phosphate synthase is rapidly induced within 15 min of ABA application, an enzyme that plays a key role in the inositol metabolism of the cell [18, 19]. The expression of the key enzyme ADP-glucose pyrophosphorylase (APL) for starch production[13] is significantly changed during turion formation. Still, not much information is known about the global transcriptome profiling for turion formation in this model system.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for altering carbon partitioning from starch to lipids in biomass produced from Duckweed cultures is disclosed. In one embodiment the method comprises introducing an agent or gene variant, which modulates expression of a gene product identified in Table S1, wherein the agent or gene variant is effective to reduce starch production and increase lipid production in said culture relative to control untreated or unaltered cultures. The agent may be a nucleic acid, a small molecule, an antibody or a chemical compound. In another embodiment, the promoters of genes with high expression levels identified in Tables S1 can either be used to overexpress coding regions of key enzymes in lipid biosynthesis or RNA interference products against transcripts of starch biosynthesis as they have been identified in Tables S1 or S2.

In one aspect of the method, the agent or gene variant inhibits or increases expression of at least one gene product identified in Table S1, the inhibition or increase resulting in increased lipid production in biomass obtained from said Duckweed culture.

In a preferred aspect of the inventive method the agent modulates, (e.g., inhibits or increases) expression of at least one gene selected from the group consisting of AGPS1, AGPL3, GBSSI, APL1, ACCase4, GPAT1, and DGAT2. Alternatively, the agent modulates production of at least one gene in Table S2, which shifts carbon partitioning, in turn increasing lipid and or protein biosynthesis.

In yet another aspect of the invention, a Duckweed plant produced from any of the methods described above is provided. In a particularly preferred embodiment, the Duckweed plant is Spirodelapolyrhiza. Also encompassed by the present invention are plant parts, progeny, seed or cells obtained from the duckweed plants described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison between frond and mature turion by TEM.A. A frond cell with a big vacuole and well-shaped chloroplasts but few and less starch granules, Bar=2 μm; B. A turion cell with thick cell wall and abundant starch granules, Bar=2 μm; Abbreviation: cell wall (CW), chloroplast (C), starch granule (S), vacuole (V) and nucleus (N).

FIG. 2. Biological variation for biological replicates from fronds and developing turions. Biological variation was represented by the square coefficient of variation of FPKM values for each gene (CV²).

FIG. 3. Comparison of APL gene expression from qRT-PCR vs. RNA-Seq. A. APL gene expression from qRT-PCR; B. APL gene expression from RNA-Seq data. Abbreviation: APL1-JN180634; APL2-JN180635; APL3-JN180636.

FIG. 4. Alignment of ABF domain from Spirodela. The amino acid sequences of bZIP protein sequence from Spirodela were aligned and the conserved regions were demonstrated here. The consensus amino acids were labeled from conserved regions and highlight as motif 1 and motif 2, the primary structure of bZIP domains (basic region and leucine zipper). All members contain these four domains except SpbZIP, which only has a basic region and a leucine zipper. SpABF1-Spipo4G0008600; SpABF2-Spipo6G0055300; SpABF3-Spipo15G0021000; SpABF4-Spipo4G0111500; SpABF5-Spipo7G0034500; SpABF6-Spipo3G0017700; SpABF7-Spipo13G0002500; SpbZIP-Spipo2G0055800.

FIG. 5. Alignment of the ERF domain from Arabidopsis and Spirodela. The bar and black arrows indicate β sheet motif, which interacts with the GCC box of target DNA. The cross-hatched box indicates the α helix. The consensus amino acids are underlined in ERF domain. The accession numbers are: AtERF1-BAA32418; AtERF2-BAA32419; AtERF3-BAA32420; AtERF4-BAA32421; AtERF5-BAA32422; SpERF1-Spipo0G0155100; SpERF2-Spipo3G0031800; SpERF3-Spipo20G0027700; SpERF4-Spipo11G0028200.

FIG. 6. A model of development of Spirodela dormancy through the signal transduction in response to environmental stimuli. Phosphorylated proteins are labeled as pink circles with a P inside. Solid lines represent direct connections. The dotted line indicates indirect connection. Not all linkages and details of pathway are shown in this diagram in order to simplify the model. Abbreviations: ABA (abscisic acid), CPK (calcium-dependent protein kinase), MAPK (mitogen-activated protein kinase), ABF (ABA-responsive element binding factor), ERF (ethylene-responsive element binding factor), HSF (heat shock transcription factor), WRKY (WRKY transcription factors), ABRE (ABA-responsive element), ERE (ethylene-responsive element).

DETAILED DESCRIPTION OF THE INVENTION

Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing an as yet an untapped source for industrial applications.

We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass.

The identification and functional annotation of differentially expressed genes opens a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops.

In another aspect of the invention we have investigated the unusual mechanism of the regulation of organellar gene expression where the translation of organellar RNAs requires the replacement of a C with a U before translation, also referred to as RNA editing. Sequence specificity is achieved through a nuclear gene family that encodes PLS-type pentatricopeptide repeat proteins (PPRs). Within monocotelydonous plants, most genomes that have been sequenced belong to the order of Commelinids, but sequencing of species of the Alismatales, Spirodela (Spirodelapolyrhiza), has providing us with a new reference for the evolution of PPR proteins, and means to modulate expression of the same to confer desirable phenotypes. We used deep sequencing of non-ribosomal total RNA to determine the number and conversion efficiency of editing sites of Spirodelaorganellar mRNA. There are 66 editing sites, of which 58 are in protein coding regions. Comparison to coconut, maize, and rice suggests that RNA editing originated from a common ancestor, but that the number of PPR genes and editing sites changed independently either by losses or gains of gene copies during evolution. Based on the expression of nuclear-encoded PPR genes and RNA editing efficiency in plastids, it appears that for ˜24% of incomplete RNA editing is the result of lower level expression of individual PPR genes. Furthermore, 37 PPR genes are differentially expressed and 11 RNA editing sites show a significant change when growth is arrested at dormancy. Thus, it appears that RNA editing is regulated through the expression of gene copies in a developmental fashion.

DEFINITIONS

The term “duckweed system” or “duckweed culture” as used herein encompasses duckweed plant cultures, duckweed fronds or immature turion cultures, duckweed suspension cultures, and duckweed protoplast cell cultures.

The term “duckweed plant culture” as used herein refers to a culture comprising mostly fully differentiated duckweed plants.

A “differentiated cell,” as used herein, is a cell having at least one phenotypic characteristic (e.g., a distinctive cell morphology or the expression of a marker nucleic acid or protein) that distinguishes it from undifferentiated cells or from cells found in other tissue types. In some embodiments, the duckweed turion culture comprises duckweed turions as described elsewhere herein.

The term “duckweed suspension culture” as used herein refers to a culture comprising dispersed duckweed cells, for example dispersed duckweed callus cells. Generally, a duckweed suspension culture will comprise both single cells and unorganized cellular aggregates of varying sizes.

The term “duckweed protoplast cell culture” refers to a culture comprising duckweed cells where at least about 50%, 60%, 70%, 80% or 90% of the duckweed cells lack a cell wall. Methods for making protoplast cells from plant cells are described, for example, in Eriksson (1995) in Plant Protoplasts, Fowke et al., eds., CRC Press, herein incorporated by reference.

The term “biological function” as used herein refers to a biological activity or property of a nucleic acid molecule or polypeptide. For example, biological functions for nucleic acid molecules include modulating biological responses, coding for polypeptides, and modulating the expression of target nucleotide sequences. Examples of biological functions for polypeptides include modulating biological responses, conferring structural properties of interest, conferring biochemical activities of interest, and conferring regulatory activities of interest. Particular, non-limiting examples of modulatory and regulatory activities include the ability to bind a substrate of interest, the ability to bind a ligand of interest, the ability to catalyze a reaction of interest, the ability to modulate a response to a plant hormone, the ability to modulate a response to a plant growth regulator, the ability to modulate a response to environmental perturbation, the ability to modulate a response to physiological perturbation, the ability to modulate a response to one or more pathogens, and the ability to modulate a response to one or more toxins.

The term “duckweed” refers to members of the family Lemnaceae. This family currently is divided into four genera and 34 species of duckweed as follows: genus Spirodela (S. polyrrhiza, S. intermedia, S. punctata); genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa. microscopica, Wa. neglecta) and genus Wolfiella (Wl. caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl. rotunda, and Wl. neotropica). Any other genera or species of Lemnaceae, if they exist, are also aspects of the present invention. Lemna species can be classified using the taxonomic scheme described by Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae—A Monograph Study GeobatanischenInstitut ETH, StiftungRubel, Zurich.

“Operably linked” as used herein in reference to nucleotide sequences refers to multiple nucleotide sequences that are placed in a functional relationship with each other. For example a promoter nucleotide sequence is operably linked to a second nucleotide sequence when it is positioned such that it can drive the transcription of the second nucleotide sequence.

“Polypeptide” refers to any monomeric or multimeric protein or peptide.

“Biologically active polypeptide” refers to a polypeptide that has the capability of performing one or more biological functions or a set of activities normally attributed to the polypeptide in a biological context. Those skilled in the art will appreciate that the term “biologically active” includes polypeptides in which the biological activity is altered as compared with the native protein (e.g., suppressed or enhanced), as long as the protein has sufficient activity to be of interest for use in industrial or chemical processes or as a therapeutic, vaccine, or diagnostics reagent. Biological activity can be determined by any method available in the art. For example, the biological activity of members of the interferon family of proteins can be determined by any of a number of methods including their interaction with interferon-specific antibodies, their ability to increase resistance to viral infection, or their ability to modulate the transcription of interferon-regulated gene targets.

“Nucleotide sequence of interest” as used herein refers to any nucleotide sequence encoding a polypeptide intended for expression in duckweed. For example, nucleotide sequences encoding therapeutic (e.g., for veterinary or medical uses) or immunogenic (e.g., for vaccination) polypeptides can be expressed using transformed duckweed according to the present invention.

The terms “expression” or “production” refer to the biosynthesis of a gene product, including the transcription, translation, and assembly of said gene product.

The term “genetically modified” as used herein refers to a plant cell or plant that is modified in its genetic information by the introduction of one or more foreign polynucleotides, and that the expression of the foreign polynucleotides leads to a phenotypic change in the plant. For example, a plant that is genetically modified to alter glycosylation is modified in its genetic information by the introduction of one or more foreign polynucleotides, where expression of the polynucleotide or polynucleotides leads to a change in glycosylation in the plant.

The transitional terms “comprising”, “consisting essentially of” and “consisting of”, when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim, an in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or materials and those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

The following methods are provided to facilitate the practice of the present invention.

Sample Preparation

Spirodelapolyrhiza 7498 was grown into half-strength Schenk and Hildebrandt basal salt mixture (Sigma, 56765) with 1% sucrose liquid medium under 6-hrs light, 8-hrs dark photoperiod. Plant tissues from four biological replicates for fronds without ABA treatment and developing turions with 3-day 10 μM ABA were collected and frozen in liquid nitrogen. 10 μg of total RNA was extracted for each sample by RNA-easy Qiagen kit with RLC buffer due to second metabolites. Ribosomal RNA was depleted with a kit from Epicenter (MRZPL116) in order to increase the coverage of other RNA classes. Vegetative fronds and turions with 14 days ABA treatment were fixed, embedded, and examined under transmission electron microscope as described[13, 20].

Library Construction and Sequence Quality Control

We started with ˜300 ngrRNA-depleted total RNA, fragmented the RNA, performed reverse transcription and size-selected the cDNA, used Emulsion PCR to amplify the complex gene libraries and prevent formation of chimeric cDNA products. All steps followed the manufacturer's guide (SOLiD™ total RNA-Seq kit). To minimize potential experimental batch effect, eight samples were barcoded, pooled, and evenly distributed into three lanes. The single-end reads with the size of 75 bp were generated with our in-house SOLiD 5500 platform. The Exact Call Chemistry (ECC) module was utilized in the sequencing run, which is an optional kit that is used to further enhance sequencing accuracy by generating reference-free bases directly. After quality trimming with score of 20, reads with a minimum length of 40 bp were saved.

Read Mapping and Quantifying Gene Expression

The remaining reads were mapped to the reference genome Spirodelapolyrhiza 7498 (GenBankAccession #ATDW01000000), which was recently sequenced, assembled, and annotated, by using TopHat 2[21] with Bowtie[22]. TopHat is a fast splice junction mapper for RNA-Seq reads. It aligns RNA-Seq reads to reference genomes using the ultra high-throughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons. Gene expression levels were normalized using fragments per kilobase of exon per million mapped reads (FPKM). Transcript abundance and deferential gene expression were calculated with Cufflinks[23]. DE genes were defined, as when their absolute value of log 2 fold change was higher than 2 and their P value was less than 0.01.

As a positive control for our measurements, we used independent data obtained in a separate study under the same induction conditions as in this study from the expression of ADP-glucose pyrophosphorylase genes with qRT-PCR [13]. As a negative control, we used northern blot data of the expression of the tur4 gene obtained in yet another study [24].

Functional Annotation and cis-Element Predictions

For each DE gene, GO annotation was obtained with the program of blast2go, which uses a blast algorithm to assign GO terms to sequences based on similarity[25]. GO enrichment was performed in two groups of gene sets, respectively, one of highly expressed transcripts in turions, the other one of highly expressed transcripts in fronds based on the whole gene set of the Spirodela genome using GOseq, which adjusts the bias from gene lengths[26]. The cis-acting regulatory DNA elements were predicted by signal scan search from PLACE database[27]. PLACE is a database of motifs found in plant cis-acting regulatory DNA elements, all from previously published reports. We dissected 1-kb regions upstream of DE genes and scanned them for potential pairs of TFs and cis-elements.

The following example is provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way.

Example I

To further uncover the regulation of gene expression as the phase switches, we took advantage of RNA deep sequencing, and compared the transcriptome between fronds and developing turions. A more comprehensive understanding of the gene repertoire and its regulation during turion formation has great potential for industrial applications including the redirection of carbon flow into higher energy products.

Calibration and Selection of Tissue Samples

A comprehensive study for turion formation has been done using abscisic acid (ABA) induction[14, 15, 17, 28, 29]. Three days after ABA induction, the Spirodelaprimordium is committed to turion development, which cannot be reversed. All primary biosynthesis of protein, mRNA and DNA are shutdown resulting in the onset of the dormant state[28]. To calibrate our growing conditions with previous investigations, we used transmission electron microscopy (TEM) to investigate different developmental stages. We chose fronds and developing turions with 3 days after ABA treatment instead of 14 days because 14-day treatment is not a key transition state and RNA purification is greatly hampered by high content of starch, but mature turions with 14-days treatment provide a more complete structural image through TEM. Turion cells have thicker cell walls, multiple smaller vacuoles and distorted plastids filled with abundant starch granules, whereas frond cells differ with having well-shaped chloroplasts consistent with previous observations (FIG. 1). Therefore, growing conditions and turion induction appear to be reproducible.

Mapping RNA-Seqreads

We used eight samples in total, with each condition having four biological replicates. To eliminate potentially technical variation from biological replicates, they were multiplexed, pooled, and sequenced with the SOLiD 5500 platform. A total of 15˜41 million quality reads per sample were generated after filtering raw reads (Table 1).

The high quality reads were mapped to chloroplast[30], mitochondria[31], and nuclear genomes[32], respectively. We could clearly divide sequence reads into these three classes. Surprisingly, there was an abundance of organelle-derived transcripts with 28˜39% of total reads. With this depth of data we could assemble sequences for complete plastid and mitochondrial transcriptomes. The high proportion of organelle reads stresses the important roles of their transcripts, provides us with their expression profiles and facilitates the phylogenetic analysis[33]. Based on the combined reads of nuclear and organelle RNAs, more than 89% of our RNA-Seq reads were mappable, attesting to the performance of the sequencing platform. It also suggests that part of previously unmapped reads in other studies remained undetected because of their organellar origin[5, 34-36]. We still found that 1˜9% of total reads were derived from ribosomal RNA, which is an indication that the protocol for the depletion of ribosomal RNA from samples was reasonably successful. Such efficiency is critical for mainly uncovering the desired transcriptome with complete coverage and in a cost-effective manner[37].

Among the total reads, 53-61% originated from nuclear DNA, lower than in other cases with about 80% of mappable sequences [34, 36]. The reason could be the method we used through ribosomal RNA removal rather than polyA selection. In case of polyA selection, organelle transcripts are automatically removed due to the lack of the polyA tail in organelle transcripts, whereas most of them were captured by our method of ribosomal RNA removal. Excluding the abundant organelle and rDNA reads, nuclear reads corresponded to 29˜72× coverage for all annotated genes (Table 1), demonstrating that the depth used in our study was sufficient to cover the Spirodela nuclear transcriptome.

Identification and Validation of Differentially Expressed Genes

Comparison of frond and developing turion samples provided us with 362 differentially expressed (DE) genes. A total of 117 had greater than 10-fold difference in mRNA levels and 208 genes were up-regulated and expressed at higher levels in developing turions than in fronds, whereas 154 genes were down-regulated, indicating lower expression in turions than in fronds (Table 2).

Previous studies had indicated that a small number of biological replicates might not be robust enough because it is impossible to know whether expression patterns are specific to individuals or are characteristic for the total population. Even for RNA deep sequencing, a sufficient number of biological replicates are still required to have confidence in the measurements[38-40]. Because two biological replicates usually are not sufficient to account for sample variability, we increased this number to four independent biological replicates. The coefficient of variation to the power of two (CV²), a normalized measure of cross-replicate variability that can be useful for evaluating the quality of RNA-Seq data, was calculated to exhibit the biological variation (FIG. 2). As expected, the data showed that the abundance of the genes varied between replicate RNA samples, especially for ones with lower FPKM values. However, with four biological replicates, which take variation within the target population into account and also counteract random technical variation[23, 41], we were very confident to assess gene expression levels with accuracy.

As another quality control, we could rely on our measurements of the 3 transcripts of ADP-glucose pyrophosphorylases (APLs) for starch synthesis[13], which were done with qRT-PCR, and compared with the RNA-Seq data. Indeed, the correlation co-efficient of 0.992 indicated that the two independent measurements were consistent and showed similar patterns: APL1 (GenBankAcession #JN180634) was highly expressed in fronds and APL3 (GenBankAcession #JN180636) showed the most abundance in developing turions. However, APL2 (GenBankAcession #JN180635) was not identified as DE gene due to only 1.5 times of difference at the time point of 0 and 3^(rd) day by the threshold value of 4 (FIG. 3). A fourth gene, tur4, provided us with a negative control from an independent study [24]. The tur4 gene has the Gene ID Spipo7G0013500 in the sequenced genome of Spirodela. Expression of this gene during turion formation was studied with Northern blot analysis. Although the tur4 gene responded to ABA treatment within hours, it appeared to return to nearly normal levels of expression thereafter. Northern blot analysis showed no induction at day 3 after ABA treatment, whereas we could still detect a 2-fold increase in tur4 expression with RNA-Seq, indicating that our method is more sensitive than Northern blot analysis. However, given both the APLs and tur4 results, we selected a cut-off for DE genes at 4-fold expressional change.

Response to ABA Stimulus

The plant hormone abscisic acid (ABA) plays a major role as a signal in seed development and plant dormancy[42, 43] and regulates many important aspects, such as the synthesis of seed storage proteins, starch and lipids[44, 45]. In Spirodela, exogenous ABA effectively triggers entry into the dormant state (turions) from growth phase (fronds)[15]. We found 25 up-regulated DE genes in response to ABA stimulus or regulation based on their GO annotation (Table 3 and S1). The pathway of ABA signal transduction and response seemed to be interwoven with enzyme metabolism (kinase, synthase, and phosphatase) and other signaling pathways (transporter, ethylene). Northern blot analysis shows that ABA rapidly up-regulates tur4 transcriptional level that encodes a peroxidase, which could stimulate turion formation and growth inhibition[24].

Growth Inhibition

Dormancy is generally defined by the lack of visible growth. The shoot apices cease active growth in perennial plants when a state of dormancy is reached. The seed dormancy is observed in seeds with a quiescent phase preventing germination. The same phenomenon was investigated for Spirodelain the presence and absence of growth. When we looked at DE genes associated with Spirodela growth by RNA-Seqdata, we found genes of histone H3 (Spipo9G0039400, Spipo0G0046100 and Spipo13G0007500) and H4 (Spipo28G0019000), ribosomal protein (Spipo1G0126300), expansins (Spipo22G0026300), aquaporins (Spipo11G0033800, Spipo17G0045100), ribulose-1,5-bisphosphate carboxylase oxygenases (RuBisCO) (Spipo19G0027700, Spipo23G0013400) for carbon fixation were down-regulated in turions (Table 4). In eukaryotic cells, DNA replication requires the synthesis of histone proteins to package newly replicated DNA into nucleosomes. Expansins are a key endogenous regulator of plant cell enlargement[46]. Aquaporins support cell growth and especially contributes to cell expansion and cell division. The gene that is highly expressed in fronds (69 times higher than in turions) is aquaporin (Spipo11G0033800) (Table 4). Over-expression of aquaporin stimulates cell growth in tobacco[47] or in Arabidopsis[48]. These results further confirm our knowledge that fronds are mainly responsible for rapid growth through actively DNA assembly, protein synthesis and carbon fixation, leading to a quick biomass increase, in comparison to the turions, where these processes are greatly decreased. Previous studies also suggested this mechanism of the turion formation by measuring DNA, RNA and protein content, which showed that DNA, protein and RNA biosynthesis were largely inhibited, resulting in the decrease of cell division, expansion and differentiation[28].

Late Embryogenesis Abundant Protein (LEA) Genes are a Valuable Marker for Dormancy

On the other hand, we found some specific mRNAs were increased in developing turions, for example LEAs. Although there were five members of LEA genes (Spipo14G0001200, Spipo5G0015500, Spipo0G0166800, Spipo1G0033500, Spipo26G0007700) with increased expression in turions, the LEA gene (Spipo0G0166800) was the most up-regulated DE gene, two other LEA genes (Spipo5G0015500 and Spipo14G0001200) were exclusively expressed in developing turions (Table 5). Indeed, the promoter of these LEA genes would be ideal to ensure expression of other coding regions exclusively in turions through transgenic approaches. Additionally, LEA was found to protect other proteins against desiccation, cold, and high salinity[49] and especially accumulates when plant seeds desiccate[50]. Given their high induction, they provide valuable markers for dormancy in general. In response to dehydration, endogenous ABA levels increased dramatically followed by induction of LEA[51]. As expected, when Spirodela fronds are destined to dormant turions triggered by ABA, desiccation is an indispensable step, in which LEA proteins play pivotal roles to preserve the cellular structures and nutrients in turions.

Genes Involved in Carbon Partitioning

Starch is the major carbon reserve in plant storage organs, and ABA has a signaling role by inducing starch biosynthetic gene expression and co-ordinate carbohydrate partitioning[52]. In our study, four genes (Spipo12G0062400, Spipo18G0038500, Spipo16G0027000 and Spipo27G0011300) (Table S1) participating in starch biosynthesis were significantly enhanced in developing turions. The qRT-PCR experiment confirmed the key enzyme of large-subunit ADP-glucose pyrophosphorylase 3 (APL3) for starch biosynthesis was highly expressed in turion development[13]. The RNA-Seq study for Landoltiapunctata also revealed gene expression involved in starch biosynthesis was up-regulated under nutrient starvation[53]. Another way to accumulate starch content is to redirect carbon flow to starch biosynthesis. We found seven genes participate in the degradation of lipids by alpha-(Spipo0G0156600, Spipo0G0180000, Spipo0G0156500, Spipo5G0040500) or beta-oxidation (Spipo0G0179100, Spipo3G0031300, Spipo1G0110400), which probably allocate carbon to starch rather than fatty acids to achieve denserturions that sink to the bottom of streams during seasons (Table S1). Previously, it has been shown that the carbon flow into seeds can be rebalanced between different macromolecules with different energy content [54]. Reallocation of carbon is critical for the improvement of oil production in novel crops in the future. In oilseed species, numerous biotechnological approaches have been carried out that were aimed to maximize the flow of carbon into oil by over-expression of enzymes of the TAG assembling network[55]. Although one might argue that turions would no longer be able to sink in water when filled with lipids, in those applications biomass would be accumulated under constant temperature.

Another way to investigate the balance of carbon partitioning can be derived from the average FPKM value (Fragments Per Kilobase of transcript per Million mapped reads) of all the key genes encoding both pathways. The genes encoding for lipid production were expressed relatively low with FPKM of 28 and 22 in fronds and turions, respectively. Therefore, the level of lipids remains low throughout development (Table S2). Given the high level of starch in turions, genes in lipid production are not induced, whereas the ones for starch biosynthesis are during turion formation, providing us with a correlation between metabolic products and the regulation of the corresponding pathways. Given this correlation, we hypothesize that we could redirect carbon flow into lipids by blocking key genes of such as AGPS1, AGPL3, GBSSI and ACCase4, GPAT1, DGAT2, and over-express transcripts of the lipid pathway (Table S2) together with turion-specific promoters, like LEAs (Spipo14G0001200, Spipo5G0015500, Spipo0G0166800)(Table 5).

Turion-Specific Pathways

We found that the transcriptome also closely links the turion phenotypic variation with thick cell wall and abundant secondary metabolites like pigment. The expressions of eight members of the UDP-glycosyltransferase superfamily (Spipo2G0010600, Spipo2G0043800, Spipo16G0044000, Spipo2G0039000, Spipo14G0034300, Spipo2G0124000, Spipo5G0014300, Spipo2G0077900) and two of the cellulose synthases (Spipo28G0017100, Spipo7G0044000) involved in cell wall biosynthesis were increased (Table S1). Three dihydroflavonolreductases (Spipo7G0010700, Spipo10G0000200, Spipo14G0054900) and one flavonoid 3′,5′-hydroxylase (Spipo0G0155000) involved in the anthocyanin pathway were up-regulated (Table S1). In addition, we found the average FPKM value for all key enzymes of lignin biosynthesis were 23 in fronds but 41 in turions, which may explain the rigidity of cell wall in turion cells to defend water pressure at the bottom of waters (Table S2).

To gain a broad overview into the biological functions for DE genes, we next performed an analysis of gene ontology (GO) enrichment (Methods). We found a total of 24 enriched pathways (p<0.01) in developing turions, whereas no enriched GO was found in fronds under the null hypothesis of the entire gene set of Spirodela (Young et al., 2010). The clustered DE genes were mainly related to response to ABA, fatty acid oxidation, and ion transportation. The GO functions of leaf senescence and cell wall modification were also highlighted (Table 6).

Transcriptional Regulation of Differentially Expressed Genes

Transcription factors (TFs) are crucial components of regulatory systems, which initiate vital changes in gene expression. Thus, we examined TF gene models and found nine TFs were significantly changed including two ABA-responsive element binding factors (bZIP, Spipo4G0008600 and Spipo2G0055800), four Ethylene-responsive element binding factors (ERFs, Spipo0G0155100, Spipo3G0031800, Spipo20G0027700 and Spipo11G0028200), two heat shock TFs (HSFs, Spipo8G0037600 and Spipo9G0002000), and one WRKY TF (Spipo8G0045500) (Table 5 and S1).

ABA-Responsive Element Binding Factor

The bZIP trancription factors regulate plant development through a basic region and a leucine zipper dimerization motif that binds to DNA[56, 57]. In the complete sequence of Spirodela genome[32], an exhaustive search of the bZIP superfamily was performed and 41 members identified. Among them, seven genes belong to the ABA-responsive element binding factors (ABFs), i.e., the bZIP superfamily group A due to their structural features with conserved regions C1-C2, basic regions, and leucine zippers (FIG. 4)[56, 58]. This group is thought to play a central role in controlling ABA-responsive gene expression in seeds and vegetative tissues via binding to ABA-responsive-elements (ABREs). For example, ABI5, one member of ABFs, induces LEA expression by binding to its promoters during seed maturation[58]. Here, all seven genes showed differentially increased expression levels, whereas only SpABF1 (Spipo4G0008600) was defined as a DE gene due to a significant change (Table 5). Noticeably, SpbZIP (Spipo2G0055800), another bZIP transcription factor, was significantly decreased in developing turions (Table 5). It shared leucine residues in the basic domain but missing other 2 conserved regions, corresponding to bZIP group I in Arabidopsis. Studies of group I genes from several species indicate that they might play a role in vascular development[56]. SpbZIP might positively regulate xylem and phloem development, too. Because both structure and function of turions are equivalent to seeds, less vascular tissue is needed in turions compared to fronds and the expression of SpbZIP is decreased accordingly. Thus, we conclude that a specific subset of bZIP transcription factors are involved in turion formation.

Other TFs Involved in ABA-Mediated Gene Expression

In addition to ABF TFs, other TFs were also identified to be involved in turion development. Ethylene-responsive element binding factors (ERFs) are transcription factors that are specific to plants. A highly conserved DNA binding domain, known as the ERF domain interacting directly with the GCC box in the ethylene-responsive-element (ERE), is the unique feature of this protein family[59] (FIG. 5). ERFs also play a role in a variety of developmental processes such as flower, seed development[60], and fruit ripening[61]. We identified 57 ERF genes in the Spirodela genome, where SpERF1 (Spipo0G0155100), SpERF2 (Spipo3G0031800), and SpERF3 (Spipo20G0027700) were significantly up-regulated and SpERF4 (Spipo11G0028200) down-regulated in response to turion development (Table 5). It had been reported that AtERF1, AtERF2, ATERF5 functioned as activators of GCC box-dependent transcription in Arabidopsis leaves, but AtERF3 and AtERF4 acted as repressors[57, 59]. It also was shown that ERF2 and ERF4 enhanced the transcription of a reporter gene in tobacco protoplasts[62]. The three highly up-regulated ERFs in Spirodelaturions should therefore play an important role in turion development.

Heat shock transcription factors (HSFs) are transcriptional activators of heat shock genes. An increasing number of studies indicated that some HSFs appeared during the maturation stage of the seed, when cell division ceased and seeds adapted to desiccation and long-term survival[63]. Here, the increased expression of two HSFs (Spipo9G0002000 and Spipo8G0037600) (Table 5) might also indicate an important function for turion desiccation and survival during long periods of winter.

WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy, and seed germination[64]. In vivo and in vitro promoter-binding studies showed that WRKY TFs could either activate or repress the expression of downstream ABFs through W-box sequences present in their promoters[65]. However, whether the Spirodela WRKY TF (Spipo8G0045500) (Table 5) is a repressor or activator needs to be further investigated.

Together, the significant changes in the expressions of ABFs, ERFs, HSF and WRKY TF reflected their obligatory regulation during turion development. Their involvement in the transition from fronds to turions and their control of spatial and temporal expression of target genes provides us also with new tools to create specialized traits through tailoring of chimeric genes.

cis-Element

Control of gene expression is achieved through the binding of transcription factors to specific cis-elements in promoter regions of target genes[66]. To predict potential pairs of TFs and cis-elements, we scanned a 1-kb region upstream of DE genes with the PLACE database[27]. We found 30 up-regulated DE genes containing the cis-element of ABA-responsive element (ABRE: YACGTGGC) and 119 with ethylene-responsive element (ERE: GCCGCC) (Table S1). These target genes of ABFs and ERFs are associated with seed dehydration (like late embryogenesis abundant proteins), regulatory transcription factor, protein kinases and phosphatases (like CPK, MAPK), carbohydrate and secondary metabolism (like cellulose synthase and stachyose synthase), and senescence-associated proteins (like Glutathione-S-transferase).

Discussion

ABA is essential for seed maturation and also enforces a period of seed dormancy so that the seeds do not germinate prematurely during unseasonably conditions. The same behavior is seen in dormant Spirodelaturions that are induced by low temperature, limited nutrition, or exogenous ABA[67]. The external stimuli rapidly induce both Ca²⁺ influx and endogenous ABA synthesis[68]. In maturing seed, ABA-regulated genes include those required for the synthesis of storage reserves and the acquisition of desiccation tolerance. Ca²⁺ can act as secondary messenger to activate the expression of cascade components of calcium-dependent protein kinase (CPK) and mitogen-activated protein kinase (MAPK). The structure of CPK shows there are four Ca²⁺-binding EF hand domains allowing the protein to function as a Ca²⁺ sensor. In addition to Ca²⁺, reversible phosphorylation also regulates kinase activity[69]. A number of studies have demonstrated that MAPKs in Arabidopsis are associated with hormone biosynthesis and signaling including ethylene and ABA[43]. Both of CPK and MAPK could phosphorylate a wide range of target proteins, including other kinases and/or transcription factors[44, 57], in particular SpERF of Spipo0G0155100, Spipo3G0031800 and Spipo20G0027700, SpABF of Spipo4G0008600 and Spipo2G0055800, SpHSF of Spipo8G0037600 and Spipo9G0002000, and SpWRKY of Spipo8G0045500 (Table 5). The activation of TFs ultimately regulates their target genes to cease cell division but begin to accumulate secondary metabolites. As shown in flowering seeds, aspects of reserve accumulation and late embryogenesis abundant (LEA) gene expression are controlled largely by the coordinated action of transcription factors[44]. Taken together, we generated a model summarizing the signal transduction leading to Spirodela dormancy based on integration of our result and previous knowledge (FIG. 6).

CONCLUSIONS

Many studies have been concerned with seed development in plants. Seeds are the product of sexual reproduction and the segregation of Mendelian traits. They also represent a dormant state in the life cycle of the plant and they compartmentalize nutrients for growth in the absence of photosynthesis. Agriculture could not exist without these properties of plants. Here, we studied a plant that propagates by clonal division and can undergo dormancy without forming seeds. The aquatic plant Spirodela could not survive on water surface without human intervention, when the water freezes. It simply switches to dormancy and accumulates starch that allows it to sink to the bottom of the water to escape the ice. Besides low temperature, however, the same switch can be achieved with the hormone ABA that has been shown to perform the same change for seed maturation. Using such an induction with Spirodela, we can study genes that regulate dormancy. Here, we isolated total RNA exclude ribosomal RNA before and at the onset of dormancy, sequenced them with next-generation technology, and identified the transcripts by mapping them back to the genome sequence. The detailed analysis of the transcriptional landscape of differentially expressed genes provides the first comprehensive view at the dormancy of aquatic plants. On the other hand, research studies have been initiated with the goal of developing duckweed species as an alternative to algae for oil production with the fact of fast growth and quick biomass accumulation[70]. The expression data for lipid and starch biosynthesis together with the turion-specific transcriptional genes from our RNA-Seq data would be the ideal targets to develop duckweeds into oil crops.

TABLE 1 Summary of sequence read alignments to three genome references. Reads # Map Qualified map nuclear nuclear Nuclear Map Map Map Sample total reads genome genome coverage chloroplast mitochondria rDNA fronds 1 24,356,014 12,795,916 53% 42 35% 1% 4% fronds 2 41,310,111 22,039,845 53% 72 37% 3% 4% fronds 3 28,333,911 16,444,539 58% 54 29% 2% 6% fronds 4 28,188,669 16,282,775 58% 53 30% 2% 9% turions 1 26,484,522 15,431,023 58% 50 28% 2% 1% turions 2 28,466,211 16,123,639 57% 53 34% 2% 2% turions 3 25,754,050 15,697,393 61% 51 26% 2% 3% turions 4 14,996,833 8,824,987 59% 29 29% 2% 1%

TABLE 2 Fold change in differentially expressed genes between in fronds and developing turions at FDR <0.01. Fold change 4.0-5.0 5.1-10 10.1-15 15.1-20 >20 Sum Genes expressed 37 73 12 10 22 154 lower in turions than fronds Genes expressed 38 97 25 15 33 208 higher in turions than fronds

TABLE 3 FPKM for Up-regulated DE genes in response to ABA stimulus. Fold Frond Turion Gene ID change FPKM FPKM Annotation Spipo6G0001100 146 0.3 45.3 Peripheral-type benzodiazepine receptor Spipo5G0029200 57 0.8 48.1 Major facilitator superfamiiy protein Spipo19G0014500 43 0.5 22.4 Galactinol synthase Spipo26G0007700 17 8.4 140.0 Late embryogenesis abundant protein LEA Spipo8G0058900 16 1.2 19.4 Flowering locus T/Terminal flower 1-like protein Spipo4G0016300 15 15.2 235.2 Annexin Spipo18G0029800 15 28.7 420.3 O-acetyltransferase-iike Spipo3G0078900 14 0.4 6.2 Stachyose synthase, putative Spipo0G0155100 13 1.7 21.5 Ethylene-responsive transcription factor 1 Spipo0G0130700 9 1.6 15.5 C4-dicarboxylate transporter Spipo7G0041900 7 1.4 9.8 ABC transporter G family member Spipo3G0031800 7 10.3 74.2 Ethylene-responsive transcription factor 2 Spipo8G0062500 6 7.2 44.3 Receptor-like protein kinase Spipo14G0026800 5 4.0 18.3 Eukaryotic aspartyl protease family protein Spipo12G0003900 4 37.9 162.9 myb domain protein 73 Spipo5G0040500 8 23.1 189.7 Alpha-dioxygenase Spipo0G0156500 6 15.5 97.9 Alpha-dioxygenase Spipo0G0180000 6 98.0 561.3 Alpha-dioxygenase Spipo0G0156600 5 19.5 104.4 Prostaglandin G/H synthase Spipo8G0046200 67 0.2 15.6 Protein phosphatase 2c, putative Spipo3G0013100 38 0.5 20.2 NAC domain-containing protein 67 Spipo23G0012800 32 1.1 34.2 Protein phosphatase 2C Spipo21G0022300 10 6.1 59.1 Protein phosphatase 2c, putative Spipo1G0021700 6 3.2 18.9 Protein phosphatase 2c, putative Spipo6G0056800 4 11.5 46.5 NAC domain-containing protein 67

TABLE 4 FPKM for Down-regulated DE genes associated with Spirodela growth. Fold Frond Turion Gene ID change FPKM FPKM Annotation Spipo11G0033800 69 33.6 0.5 Aquaporin Spipo17G0045100 5 86.3 17.8 Aquaporin Spipo22G0025300 5 186.4 40.4 Expansin Spipo9G0039400 7 68.2 9.4 Histone H3 Spipo0G0046100 7 112.4 16.4 Histone H3 Spipo13G0007500 6 159.5 27.5 Histone H3 Spipo28G0019000 5 77.9 14.8 Histone H4 Spipo3G0024800 14 1018.4 71.2 Pre-rRNA-processing protein PNO1 Spipo1G0126300 5 1371.4 293.1 60S ribosomal protein L10-like protein Spipo19G0027700 29 6951.7 241.3 Ribulose bisphosphate carboxylase small chain Spipo23G0013400 5 476.1 93.1 Ribulose-1 5- bisphosphate carboxylase/oxygenase activase

TABLE 5 FPKM for Turion-specific genes and DE transcriptional factors. Fold Frond Turion Gene ID change FPKM FPKM Directionality Annotation Spipo14G0001200 NA 0.0 31.0 up-regulated Late embryogenesis abundant protein LEA Spipo5G0015500 NA 0.0 45.8 up-regulated Late embryogenesis abundant protein LEA Spipo0G0166800 170 1.4 235.2 up-regulated Late embryogenesis abundant protein LEA Spipo1G0033500 34 3.4 114.8 up-regulated Late embryogenesis abundant protein LEA Spipo26G0007700 17 8.4 140.0 up-regulated Late embryogenesis abundant protein LEA Spipo4G0008600 5 6.1 33.1 up-regulated bZIP transcription factor A Spipo8G0037600 11 1.1 11.6 up-regulated Heat shock transcription factor A2 Spipo9G0002000 5 14.2 67.8 up-regulated Heat shock transcription factor A2 Spipo0G0155100 13 1.7 21.5 up-regulated Ethylene-responsive transcription factor 1 Spipo3G0031800 7 10.3 74.2 up-regulated Ethylene-responsive transcription factor 2 Spipo20G0027700 5 10.6 53.1 up-regulated Ethylene-responsive transcription factor 3 Spipo11G0028200 7 32.7 4.4 down-regulated Ethylene-responsive transcription factor 4 Spipo8G0045500 7 11.3 1.7 down-regulated WRKY transcription factor, putative Spipo2G0055800 4 17.1 4.0 down-regulated bZIP transcription factor I

TABLE 6 Functional GO enrichment in developing turions. Enriched GO ID description GO: 0001561 fatty acid alpha-oxidation GO: 0033539 fatty acid beta-oxidation using acyl-CoA dehydrogenase GO: 0010167 response to nitrate GO: 0015706 nitrate transport GO: 0055114 oxidation-reduction process GO: 0009830 cell wall modification involved in abscission GO: 0009651 response to salt stress GO: 0010106 cellular response to iron ion starvation GO: 0010150 leaf senescence GO: 0009737 response to abscisic acid stimulus GO: 0006826 iron ion transport GO: 0001676 long-chain fatty acid metabolic process GO: 0001666 response to hypoxia GO: 0046487 glyoxylate metabolic process GO: 0071732 cellular response to nitric oxide GO: 0010286 heat acclimation GO: 0071446 cellular response to salicylic acid stimulus GO: 0072329 monocarboxylic acid catabolic process GO: 0019579 aldaric acid catabolic process GO: 0009751 response to salicylic acid stimulus GO: 0042542 response to hydrogen peroxide GO: 0046686 response to cadmium ion GO: 0009788 negative regulation of abscisic acid mediated signaling pathway GO: 0009414 response to water deprivation

ABBREVIATIONS

ABA: abscisic acid; FPKM: Fragments Per Kilobase of transcript per Million mapped reads; DE gene: differentially expressed genes; LEA: late embryogenesis abundant protein; ABF: ABA-responsive element binding factors; ERF: Ethylene-responsive element binding factors; CPK: calcium-dependent protein kinase; MAPK: mitogen-activated protein kinase; CCR: Cinnamoyl-CoA reductase; CAD: cinnamyl-alcohol dehydrogenase; APS: ADP-glucose pyrophosphorylase small subunit; APL: ADP-glucose pyrophosphorylase large subunit; SS: starch synthase; GBSS: Granule-bound starch synthase; BE: Starch branching enzyme; DBE: Starchdebranching enzyme; ACCase: Acetyl-CoA carboxylase; GPAT: Glycerol-3-phosphate acyltransferase; AGPAT: Acylglycerophosphateacyltransferase; DGAT: Diacylglycerolacyltransferase.

TABLE S1 Fold Frond Turion Direction- Gene ID change FPKM FPKM ality Annotation ABRE ERE GO Function Spipo14G0001200 NA 0.0 31.0 up-regulated Late embryogenesis abundant NO YES protein LEA Spipo5G0015500 NA 0.0 45.8 up-regulated Late embryogenesis abundant YES YES protein LEA Spipo0G0166800 170 1.4 235.2 up-regulated Late embryogenesis abundant NO NO protein LEA Spipo6G0001100 146 0.3 45.3 up-regulated Peripheral-type benzodiazepine NO YES GO: 0009737 response to receptor abscisic acid stimulus Spipo10G0013800 99 0.1 9.5 up-regulated 70 kDa heat shock protein NO YES Spipo21G0043200 97 0.7 66.3 up-regulated Protein of unknown function, NO YES DUF599 Spipo25G0008400 95 0.2 18.0 up-regulated myb domain protein 73 NO NO Spipo2G0010600 84 0.2 19.7 up-regulated UDP-Glycosyltransferase NO NO superfamily Spipo2G0035100 72 0.3 18.2 up-regulated Unknown protein NO YES Spipo1G0017700 71 0.5 34.3 up-regulated (R)-limonene synthase, putative NO NO Spipo8G0046200 67 0.2 15.6 up-regulated Protein phosphatase 2c, putative YES YES GO: 0009788 negative regulation of abscisic acid mediated signaling pathway Spipo13G0026700 58 4.9 285.5 up-regulated Sulfite oxidase NO NO Spipo5G0029200 57 0.8 48.1 up-regulated Major facilitator superfamily protein NO NO GO: 0009737 response to abscisic acid stimulus Spipo2G0043800 54 6.4 340.8 up-regulated UDP-Glycosyltransferase superfamily NO YES Spipo16G0044000 52 0.2 8.8 up-regulated UDP-Glycosyltransferase superfamily NO YES Spipo11G0050900 49 20.2 986.4 up-regulated Kunitz trypsin inhibitor NO YES Spipo19G0014500 43 0.5 22.4 up-regulated Galactinol synthase NO NO GO: 0009737 response to abscisic acid stimulus Spipo4G0003000 42 0.5 22.8 up-regulated Glutamate decarboxylase, putative NO YES Spipo25G0001000 42 1.0 42.4 up-regulated Exostosin family protein NO YES Spipo14G0016400 41 0.8 33.7 up-regulated Lachrymatory-factor synthase NO NO Spipo3G0013100 38 0.5 20.2 up-regulated NAC domain-containing protein 67 NO NO GO: 0009788 negative regulation of abscisic acid mediated signaling pathway Spipo2G0039000 38 0.4 13.6 up-regulated UDP-Glycosyltransferase superfamily NO NO protein Spipo1G0033500 34 3.4 114.8 up-regulated Late embryogenesis abundant NO YES protein LEA Spipo15G0020300 34 1.5 52.2 up-regulated Cytochrome P450 NO NO Spipo12G0048100 33 0.0 1.6 up-regulated Pentatricopeptide repeat-containing NO YES protein, putative Spipo10G0053200 33 0.1 3.6 up-regulated Sucrose phosphate synthase NO NO Spipo23G0012800 32 1.1 34.2 up-regulated Protein phosphatase 2C NO YES GO: 0009788 negative regulation of abscisic acid mediated signaling pathway Spipo30G0012600 31 2.8 89.1 up-regulated Unknown protein NO YES Spipo9G0021500 29 0.7 19.2 up-regulated Nucleotide-sugar transporter family YES NO protein Spipo12G0044900 29 0.4 11.1 up-regulated CBL-interacting protein kinase 9 YES YES Spipo0G0148600 27 23.1 635.9 up-regulated Unknown protein NO NO Spipo15G0047700 27 0.1 1.6 up-regulated Cytochrome P450 NO YES Spipo0G0175800 2.1 1.4 28.2 up-regulated EC metallothionein-like protein NO YES Spipo0G0138500 19 10.6 206.5 up-regulated Unknown protein YES YES Spipo4G0033500 19 0.1 1.0 up-regulated Pentatricopeptide repeat-containing NO NO protein, putative Spipo2G0012200 19 0.4 7.8 up-regulated Glutamate dehydrogenase NO YES Spipo4G0112100 19 1.6 31.2 up-reglated 9-cis-epoxycarotenoid dioxygenase 1 NO NO Spipo1G0101000 18 0.1 1.6 up-regulated Pentatricopeptide repeat-containing NO YES protein Spipo21G0039100 18 12.7 232.4 up-regulated Unknown protein NO NO Spipo26G0007700 17 8.4 140.0 up-regulated Late embryogenesis abundant YES YES GO: 0009737 response to protein LEA abscisic acid stimulus Spipo10G0023800 17 0.8 13.8 up-regulated Class 1 heat shock protein NO NO Spipo14G0034300 16 3.9 62.6 up-regulated UDP glycosyltransferase YES NO Spipo9G0006300 16 2.1 32.6 up-regulated Phosphoserine aminotransferase NO NO Spipo8G0058900 16 1.2 19.4 up-regulated Flowering locus T/Terminal flower NO YES GO: 0009737 response to 1-like protein abscisic acid stimulus Spipo1G0068300 15 8.6 133.2 up-regulated Heavy metal transport/detoxification YES YES superfamily protein Spipo4G0016300 15 15.2 235.2 up-regulated Annexin NO NO GO: 0009737 response to abscisic acid stimulus Spipo2G0064500 15 3.6 56.0 up-regulated Cytochrome P450 NO YES Spipo6G0047600 15 0.7 10.1 up-regulated Beta glucosidase like protein NO NO Spipo28G0015900 15 0.1 1.6 up-regulated Trehalose-6-phosphate synthase NO YES Spipo18G0029800 15 28.7 420.3 up-regulated O-acetyltransferase-like NO NO GO: 0009737 response to abscisic acid stimulus Spipo1G0076000 14 0.6 8.6 up-regulated Putative lysine decarboxylase family NO NO protein Spipo8G0061900 14 4.6 64.7 up-regulated LOB domain-containing protein, YES YES putative Spipo11G0038800 14 0.9 13.4 up-regulated Arabidopsis protein of unknown NO YES function (DUF241) Spipo3G0078900 14 0.4 6.2 up-regulated Stachyose synthase, putative YES NO GO: 0009737 response to abscisic acid stimulus Spipo18G0020700 14 10.6 143.8 up-regulated Formate dehydrogenase NO NO Spipo4G0080100 13 0.2 2.3 up-regulated Cytochrome P450, putative NO YES Spipo0G0155100 13 1.7 21.5 up-regulated Ethylene-responsive transcription YES YES GO: 0009737 response to factor 1 abscisic acid stimulus Spipo28G0001000 13 3.3 42.3 up-regulated Phosphofructokinase, putative NO YES Spipo15G0037300 13 3.8 48.6 up-regulated Mitogen-activated protein kinase NO YES kinase 1 (MAPK) Spipo2G0067900 12 0.3 4.0 up-regulated Heat shock 70 kDa protein 1 NO YES Spipo26G0000300 12 6.6 79.8 up-regulated unknown protein NO YES Spipo2G0124000 12 0.6 7.7 up-regulated UDP-Glycosyltransferase superfamily NO NO protein Spipo4G0079600 12 30.7 364.1 up-regulated 12-oxophytodienoate reductase NO YES Spipo2G0096800 12 13.9 160.7 up-regulated unknown protein NO YES Spipo1G0074600 11 22.6 257.1 up-regulated Unknown protein NO YES Spipo7G0045000 11 12.2 137.5 up-regulated Sucrose phosphate synthase NO YES Spipo8G0037600 11 1.1 11.6 up-regulated heat shock transcription factor A2 NO YES Spipo21G0039300 11 3.2 35.0 up-regulated Unknown protein NO NO Spipo5G0014300 11 3.4 36.1 up-regulated UDP-Glycosyltransferase superfamily NO YES protein Spipo28G0003500 11 30.5 322.2 up-regulated Gibberellin-regulated protein NO YES Spipo1G0058300 10 0.1 1.0 up-regulated Pentatricopeptide repeat-containing NO YES protein Spipo3G0032200 10 10.4 105.2 up-regulated Epoxide hydrolase 1 NO YES Spipo0G0116100 10 6.2 62.5 up-regulated Beta-glucosidase, putative NO YES Spipo4G0092300 10 45.1 447.5 up-regulated Alcohol dehydrogenase NO YES Spipo21G0022300 10 6.1 59.1 up-regulated Protein phosphatase 2c, putative NO YES GO: 0009788 negative regulation of abscisic acid mediated signaling pathway Spipo19G0036000 10 1.2 11.2 up-regulated Aspartic proteinase, putative NO NO Spipo19G0016500 10 2.8 27.3 up-regulated Isocitrate dehydrogenase [NADP] NO NO Spipo0G0130700 9 1.6 15.5 up-regulated C4-dicarboxylate transporter/malic NO NO GO: 0009737 response to acid transport protein abscisic acid stimulus Spipo7G0066600 9 164.7 1539.8 up-regulated Glyceraldehyde-3-phosphate YES YES dehydrogenase Spipo22G0037200 9 18.8 173.7 up-regulated Amino acid selective channel protein NO NO Spipo6G0047400 9 5.3 47.4 up-regulated Universal stress protein NO NO Spipo25G0008100 9 1.5 13.5 up-regulated Alpha-mannosidase-like protein NO YES Spipo5G0050700 9 0.6 5.3 up-regulated Sugar phosphate exchanger 2 NO NO Spipo24G0000900 9 0.3 2.9 up-regulated Kinase, putative NO NO Spipo2G0047500 8 17.8 148.0 up-regulated SAUR-like auxin-responsive protein YES YES family Spipo11G0001500 8 22.3 185.3 up-regulated Abscisic acid 8′-hydroxylase 3 NO NO Spipo5G0064900 8 10.7 88.2 up-regulated Tetratricopeptide repeat (TPR)-like NO YES superfamily protein Spipo5G0040500 8 23.1 189.7 up-regulated Alpha-dioxygenase NO NO GO: 0009737; response to GO: 0001561 abscisic acid stimulus; fatty acid alpha- oxidation Spipo27G0011300 8 13.8 112.1 up-regulated Beta-glucosidase A NO NO GO: 0019252 starch biosynthetic process Spipo1G0069600 8 32.9 266.4 up-regulated 26S proteasome regulatory subunit NO YES S3, putative Spipo11G0031500 8 3.9 31.8 up-regulated Cytochrome P450, putative NO NO Spipo2G0040400 8 0.8 6.1 up-regulated Cytochrome P450 YES YES Spipo11G0039200 8 5.8 45.4 up-regulated Alpha-amylase, putative NO YES Spipo3G0041300 8 0.2 1.3 up-regulated Pentatricopeptide repeat-containing NO YES protein Spipo14G0010200 8 1.8 13.9 up-regulated Protein CHUP1 NO YES Spipo11G0060400 8 1.5 11.7 up-regulated Folate/biopterin transporter family NO YES protein Spipo5G0023500 8 6.1 47.4 up-regulated Protein of unknown function NO NO (DUF1262) Spipo1G0032700 8 11.8 91.9 up-regulated 2-succinylbenzoate-CoA ligase NO NO Spipo16G0027000 8 4.4 33.4 up-regulated Glutathione-S-transferase (GST) NO YES GO: 0019252 starch biosynthetic process Spipo0G0176700 8 0.6 4.9 up-regulated Ovate family protein 1 NO YES Spipo18G0038500 8 36.0 291.5 up-regulated Glucose-1-phosphate NO NO GO: 0019252 starch adenylyltransferase biosynthetic process Spipo11G0025200 7 2.1 15.6 up-regulated methionine gamma-lyase NO YES Spipo13G0053800 7 6.9 51.6 up-regulated Glyceraldehyde 3-phosphate YES NO dehydrogenase Spipo5G0077000 7 0.4 2.7 up-regulated Pentatricopeptide repeat-containing NO NO protein Spipo23G0007500 7 1.5 10.9 up-regulated O-fucosyltransferase family protein NO NO Spipo5G0014900 7 1.0 7.1 up-regulated Cytochrome P450, putative YES YES Spipo29G0009400 7 3.4 24.9 up-regulated Dof zinc finger protein NO NO Spipo7G0041900 7 1.4 9.8 up-regulated ABC transporter G family member NO YES GO: 0009737 response to abscisic acid stimulus Spipo3G0031800 7 10.3 74.2 up-regulated Ethylene-responsive transcription NO NO GO: 0009737 response to factor 2 abscisic acid stimulus Spipo21G0009000 7 4.7 33.4 up-regulated Auxin responsive protein YES YES Spipo0G0124700 7 2.0 14.1 up-regulated G-type lectin S-receptor-like NO YES serine/threonine-protein kinase Spipo19G0037600 7 21.2 144.8 up-regulated Hydroxyacylglutathione hydrolase NO YES Spipo3G0073100 7 14.3 96.6 up-regulated Peroxiredoxin NO YES Spipo22G0042300 7 24.8 166.4 up-regulated Soul heme-binding family protein NO YES Spipo10G0025700 7 1.1 7.6 up-regulated Histidine decarboxylase NO NO Spipo7G0061700 7 140.8 936.4 up-regulated Cysteine-rich secretory protein NO YES Spipo18G0031500 7 2.8 18.8 up-regulated LOB domain-containing protein 41 NO YES Spipo8G0027700 7 1.4 8.9 up-regulated MATE efflux family protein YES NO Spipo28G0017100 7 1.6 10.6 up-regulated Cellulose-synthase-like C5 NO YES Spipo0G0155000 6 1.5 9.8 up-regulated Cytochrome P450 flavonoid 3′,5′- NO NO hydroxylase Spipo7G0010700 6 10.1 64.8 up-regulated Dihydroflavonol 4-reductase NO YES Spipo10G0000200 6 26.2 167.8 up-regulated Dihydroflavonol 4-reductase NO NO Spipo15G0031400 6 3.3 21.1 up-regulated Major facilitator superfamily protein NO YES Spipo0G0156500 6 15.5 97.9 up-regulated Alpha-dioxygenase NO NO GO: 0009737; response to GO: 0001561 abscisic acid stimulus; fatty acid alpha- oxidation Spipo1G0110400 6 5.8 36.8 up-regulated Electron transfer flavoprotein- NO NO GO: 0033539 fatty acid ubiquinone oxidoreductase beta- oxidation Spipo6G0053900 6 6.5 39.9 up-regulated Major facilitator superfamily NO NO antiporter, putative, expressed Spipo8G0062500 6 7.2 44.3 up-regulated Receptor-like protein kinase NO YES GO: 0009737 response to abscisic acid stimulus Spipo14G0009000 6 1.7 10.6 up-regulated Major facilitator superfamily protein NO NO Spipo15G0028800 6 2.5 15.1 up-regulated Cysteine-rich receptor-like protein NO YES kinase Spipo28G0011200 6 2.3 13.5 up-regulated Chaperone clpb, putative NO NO Spipo5G0051100 6 7.8 46.5 up-regulated Cytochrome P450, putative NO YES Spipo11G0020200 6 116.8 695.6 up-regulated Kunitz trypsin inhibitor 4 NO NO Spipo1G0021700 6 3.2 18.9 up-regulated Protein phosphatase 2c, putative NO YES GO: 0009788 negative regulation of abscisic acid mediated signaling pathway Spipo7G0059600 6 28.6 168.7 up-regulated AP-1 complex subunit gamma-2, NO NO putative Spipo3G0031300 6 16.3 95.9 up-regulated Acyl-CoA dehydrogenase NO YES GO: 0033539 fatty acid beta- oxidation Spipo26G0019100 6 3.1 17.7 up-regulated Integral membrane protein TmpA NO YES Spipo8G0065700 6 1.2 6.9 up-regulated Core-2/I-branching beta-1,6-N- NO NO acetylglucosaminyltransferase family protein Spipo17G0009300 6 1.5 8.7 up-regulated CBS domain-containing protein, YES YES putative, expressed Spipo10G0038500 6 2.1 12.1 up-regulated Phosphatidylinositol kinase family- NO YES like protein Spipo0G0180000 6 98.0 561.3 up-regulated Alpha-dioxygenase NO NO GO: 0009737; response to GO: 0001561 abscisic acid stimulus; fatty acid alpha- oxidation Spipo24G0001200 6 5.6 31.8 up-regulated Adiponectin receptor protein NO NO Spipo10G0030200 6 11.2 63.7 up-regulated Chaperone DnaJ-domain superfamily YES YES protein Spipo8G0040300 6 1.9 10.8 up-regulated 3-hydroxyisobutyrate dehydrogenase NO NO Spipo23G0017700 6 1.6 9.0 up-regulated GTPaseobg NO NO Spipo0G0148500 6 34.3 192.2 up-regulated Unknown protein NO YES Spipo16G0046000 5 17.7 96.7 up-regulated Beta-amylase YES YES Spipo3G0073400 5 5.0 27.4 up-regulated tolB protein-related NO NO Spipo4G0008600 5 6.1 33.1 up-regulated bZIP transcription factor A NO NO Spipo3G0064800 5 14.2 76.7 up-regulated Beta-1,3-glucanase NO NO Spipo0G0156600 5 19.5 104.4 up-regulated Prostaglandin G/H synthase NO NO GO: 0009737; response to GO: 0001561 abscisic acid stimulus; fatty acid alpha- oxidation Spipo11G0043800 5 10.3 54.6 up-regulated Calcium-binding EF hand family NO YES protein Spipo23G0006300 5 7.3 38.4 up-regulated RNA-binding protein YES YES Spipo12G0062400 5 2.8 14.6 up-regulated Glycogen debranching enzyme NO YES GO: 0019252 starch biosynthetic process Spipo18G0028400 5 0.8 3.9 up-regulated NHL domain-containing protein NO YES Spipo7G0060200 5 8.5 44.3 up-regulated Unknown protein NO YES Spipo10G0017900 5 5.8 30.4 up-regulated ABC transporter G family member NO YES Spipo2G0099700 5 1.4 7.5 up-regulated unknown protein NO NO Spipo11G0031600 5 2.9 15.1 up-regulated Cytochrome P450 NO NO Spipo16G0030600 5 4.1 21.5 up-regulated Glutamine synthetase NO NO Spipo2G0061100 5 3.4 17.5 up-regulated Prolyl 4-hydroxylase alpha subunit, NO YES putative Spipo20G0017000 5 4.5 22.9 up-regulated Isoflavonereductase-like protein NO NO Spipo4G0013100 5 55.2 282.1 up-regulated Inositol-3-phosphate synthase NO YES Spipo4G0033200 5 18.5 94.4 up-regulated MtN19-like protein NO YES Spipo14G0054900 5 2.0 9.9 up-regulated Dihydroflavonol 4-reductase NO YES Spipo2G0071700 5 0.4 1.9 up-regulated Pentatricopeptide repeat-containing NO YES protein, putative Spipo0G0030300 5 47.3 238.4 up-regulated Aldehyde dehydrogenase YES YES Spipo3G0002000 5 1.6 7.8 up-regulated Xanthine/uracil permease family YES YES protein Spipo20G0027700 5 10.6 53.1 up-regulated Ethylene-responsive transcription NO NO factor 3 Spipo7G0013600 5 8.2 40.9 up-regulated 2-oxoglutarate (2OG) and Fe(II)- NO NO dependent oxygenase superfamily Spipo0G0013700 5 2.4 12.0 up-regulated Unknown protein YES YES Spipo2G0018300 5 9.6 47.7 up-regulated Early light-induced protein NO NO Spipo7G0048800 5 16.7 82.8 up-regulated NAC domain protein NO YES Spipo8G0040000 5 1.2 5.5 up-regulated 2-hydroxy-3-oxopropionate reductase NO YES Spipo9G0002000 5 14.2 67.8 up-regulated Heat shock transcription factor A-2 NO NO Spipo9G0033600 5 123.3 585.4 up-regulated Fructose-bisphosphatealdolase NO YES Spipo23G0001600 5 3.2 15.2 up-regulated Kinase, pfkB family protein, NO YES expressed Spipo7G0044000 5 4.4 20.5 up-regulated Cellulose synthase-like B3 YES YES Spipo6G0053800 5 3.7 17.3 up-regulated Major facilitator superfamily NO NO antiporter, putative, expressed Spipo2G0077900 5 6.1 28.4 up-regulated UDP-Glycosyltransferase superfamily NO NO Spipo14G0026800 5 4.0 18.3 up-regulated Eukaryotic aspartyl protease family NO YES GO: 0009737 response to protein abscisic acid stimulus Spipo0G0179100 5 11.5 53.2 up-regulated Electron transfer flavoprotein- NO YES GO: 0033539 fatty acid ubiquinone oxidoreductase beta- oxidation Spipo1G0003700 5 7.6 35.1 up-regulated Calcium-dependent protein kinase YES NO (CPK) Spipo15G0048400 5 7.7 34.9 up-regulated Cathepsin L-like cysteine proteinase NO YES Spipo0G0154800 5 4.4 19.7 up-regulated Xanthine dehydrogenase/oxidase NO YES Spipo2G0033500 4 7.7 34.6 up-regulated Lysine- NO NO ketoglutaratereductase/saccharopine dehydrogenase Spipo4G0038800 4 3.9 17.3 up-regulated GDSL esterase/lipase NO NO Spipo13G0039800 4 16.2 70.6 up-regulated Cytochrome P450 NO NO Spipo3G0073500 4 2.9 12.4 up-regulated tolB protein-related NO NO Spipo7G0066500 4 1.6 6.9 up-regulated Multidrug resistance protein ABC YES YES transporter family Spipo24G0015900 4 2.8 12.2 up-regulated Unknown protein NO NO Spipo9G0002200 4 105.3 452.4 up-regulated Thioredoxin NO YES Spipo12G0003900 4 37.9 162.9 up-regulated Myb domain protein 73 NO YES GO: 0009737 response to abscisic acid stimulus Spipo2G0096700 4 55.9 238.4 up-regulated Peroxidase 73, putative NO YES Spipo26G0017500 4 5.7 24.1 up-regulated Exostosin family protein NO YES Spipo7G0028500 4 23.1 97.9 up-regulated Potassium transporter 11 NO NO Spipo5G0012100 4 17.9 75.8 up-regulated CCR4-NOT transcription complex NO YES subunit 7 Spipo19G0030800 4 6.0 25.1 up-regulated Inositol-pentakisphosphate 2-kinase, NO NO putative Spipo20G0012400 4 1.5 6.2 up-regulated RING/U-box superfamily protein NO YES Spipo23G0021500 4 5.4 22.8 up-regulated SEC14 cytosolic factor family NO YES protein/phosphoglyceride transfer family protein Spipo22G0012700 4 31.1 130.2 up-regulated Rubber elongation factor protein NO YES (REF) Spipo18G0007900 4 70.2 289.4 up-regulated Aspartate aminotransferase NO YES Spipo0G0085700 4 14.1 57.9 up-regulated Unknown protein NO NO Spipo16G0033600 4 2.5 10.2 up-regulated Alpha-galactosidase 1 YES NO Spipo3G0022400 4 11.3 46.0 up-regulated Glutathione-S-transferase (GST) NO YES Spipo6G0056800 4 11.5 46.5 up-regulated NAC domain-containing protein 67 YES YES GO: 0009788 negative regulation of abscisic acid mediated signaling pathway Spipo3G0033300 4 8.9 35.9 up-regulated Protein of unknown function, NO NO DUF538 Spipo5G0073900 4 9.2 37.0 up-regulated Alternative oxidase NO YES Spipo12G0023200 4 0.5 1.9 up-regulated Disease resistance protein NO NO Spipo11G0033800 69 33.6 0.5 down- Aquaporin NO NO regulated Spipo17G0047900 66 #### 1921.3 down- Chitobiosyldiphosphodolichol beta- YES YES regulated mannosyltransferase Spipo1G0071900 54 #### 1538.8 down- Chitobiosyldiphosphodolichol beta- NO YES regulated mannosyltransferase Spipo13G0047300 50 608.3 12.1 down- Ycf68 protein NO NO regulated Spipo12G0026200 50 8.6 0.2 down- Eukaryotic aspartyl protease family YES YES regulated protein Spipo1G0120500 45 72.3 1.6 down- Lectin NO YES regulated Spipo0G0129700 38 103.0 2.7 down- Bifunctional inhibitor/lipid-transfer NO YES regulated protein/seed storage 2S albumin superfamily Spipo1G0120400 37 27.1 0.7 down- Lectin NO NO regulated Spipo26G0006100 36 27.7 0.8 down- BURP domain-containing protein NO NO regulated Spipo1G0108500 35 44.8 1.3 down- Unknown protein NO NO regulated Spipo14G0020800 30 498.4 16.6 down- Arabinogalactan peptide 20-like YES YES regulated Spipo19G0027700 29 #### 241.3 down- Ribulosebisphosphate carboxylase NO NO regulated small chain Spipo21G0033600 27 #### 487.3 down- Oxidative stress 3 NO NO regulated Spipo1G0121700 26 1530.8 59.8 down- Bifunctional inhibitor/lipid-transfer NO NO regulated protein/seed storage 2S albumin superfamily Spipo23G0012000 25 6197.7 243.6 down- Unknown protein NO YES regulated Spipo0G0022100 24 51.0 2.1 down- Protein of unknown function, NO NO regulated DUF538 Spipo7G0031300 24 184.7 7.7 down- Unknown protein NO NO regulated Spipo6G0029000 22 15.6 0.7 down- O-acyltransferase WSD1 YES NO regulated Spipo0G0067700 22 309.9 14.0 down- Unknown protein NO YES regulated Spipo2G0110300 22 28.5 1.3 down- Cysteine-rich secretory protein NO NO regulated Spipo1G0078600 21 19.1 0.9 down- Peroxidase NO NO regulated Spipo12G0010100 20 7.7 0.4 down- Nucleobaseascorbate transporter NO NO regulated Spipo18G0014800 20 95.2 4.9 down- FASCICLIN-like arabinogalactan 6 NO YES regulated Spipo6G0002600 19 503.1 26.1 down- Unknown protein NO NO regulated Spipo3G0074100 19 11.9 0.6 down- fucosyltransferase 1 NO YES regulated Spipo5G0036600 19 1.7 0.1 down- Receptor kinase NO YES regulated Spipo10G0007200 19 7698.9 414.6 down- Unknown protein NO NO regulated Spipo11G0017100 18 19.8 1.1 down- Blue copper protein NO YES regulated Spipo8G0067000 17 11.2 0.7 down- Unknown protein YES YES regulated Spipo14G0059300 17 323.8 19.5 down- Bifunctional inhibitor/lipid-transfer NO NO regulated protein/seed storage 2S albumin-like protein Spipo1G0007400 16 258.6 16.6 down- Unknown protein NO NO regulated Spipo0G0159700 15 108.2 7.2 down- BURP domain-containing protein NO NO regulated Spipo3G0024800 14 1018.4 71.2 down- Pre-rRNA-processing protein PNO1 NO NO regulated Spipo9G0045500 14 30.0 2.2 down- Carboxyvinyl- NO NO regulated carboxyphosphonate- phosphorylmutase, putative, expressed Spipo9G0031000 13 5.1 0.4 down- Kinase, putative NO YES regulated Spipo5G0017800 13 1348.5 105.8 down- Unknown protein NO NO regulated Spipo8G0062200 12 11.6 0.9 down- Protein kinase family protein NO YES regulated Spipo0G0025800 12 5.8 0.5 down- Leucine-rich repeat protein kinase- NO NO regulated like protein Spipo26G0006600 12 68.2 5.9 down- BURP domain-containing protein YES NO regulated Spipo20G0016500 11 64.9 5.8 down- Plant cadmium resistance 2 NO YES regulated Spipo7G0011900 11 33.1 3.1 down- Early nodulin 20, putative NO NO regulated Spipo20G0037200 11 #### 1037.5 down- Unknown protein NO YES regulated Spipo30G0013100 10 3.4 0.3 down- Protein kinase family protein NO YES regulated Spipo8G0009900 10 15.2 1.5 down- Peroxidase 39 NO NO regulated Spipo0G0134100 10 4.7 0.5 down- Protein kinase family protein NO NO regulated Spipo2G0109900 10 141.9 14.4 down- CAP (Cysteine-rich secretory proteins NO NO regulated and Pathogenesis-related 1 protein) Spipo0G0190200 10 486.5 49.4 down- Purple acid phosphatase 1 NO NO regulated Spipo10G0029700 10 2.4 0.2 down- Disease resistance gene homolog 9N NO NO regulated Spipo30G0015100 9 7.6 0.8 down- 9-cis-epoxycarotenoid dioxygenase 1 NO YES regulated Spipo28G0025100 9 21.4 2.3 down- Cation transport regulator-like protein YES YES regulated Spipo2G0091100 9 7.1 0.8 down- Unknown protein NO YES regulated Spipo2G0100400 9 223.5 25.3 down- Nucleotide-sugar transporter family YES YES regulated protein Spipo2G0110400 9 218.2 24.7 down- Cysteine-rich secretory protein NO YES regulated Spipo5G0027100 9 18.7 2.1 down- Myb domain protein 73 YES YES regulated Spipo1G0079200 9 14.9 1.7 down- Peroxidase NO NO regulated Spipo14G0014700 9 #### 1293.9 down- Unknown protein NO YES regulated Spipo4G0110100 9 58.4 6.8 down- Fructose-1,6-bisphosphatase class 1 YES YES regulated Spipo19G0016700 8 1666.6 196.3 down- Unknown protein NO YES regulated Spipo11G0063600 8 168.4 20.3 down- Unknown protein NO YES regulated Spipo9G0030500 8 3.8 0.5 down- Kinase, putative NO YES regulated Spipo14G0033900 8 5094.7 626.7 down- Carbonic anhydrase NO YES regulated Spipo1G0023600 8 6.2 0.8 down- HXXXD-type acyl-transferase family NO NO regulated protein Spipo1G0018600 8 52.1 6.6 down- RING finger and CHY zinc finger NO NO regulated domain-containing protein 1 Spipo3G0031100 8 47.7 6.1 down- Pectinesterase NO NO regulated Spipo7G0045100 8 855.2 109.7 down- Unknown protein NO NO regulated Spipo13G0020200 8 433.6 56.5 down- Phosphoglycolate phosphatase YES YES regulated Spipo0G0001400 8 4.2 0.6 down- Cationic amino acid transporter NO YES regulated Spipo20G0013200 8 117.3 15.6 down- Acid phosphatase, putative, expressed NO YES regulated Spipo3G0049000 7 127.9 18.6 down- Glucose-1-phosphate NO NO regulated adenylyltransferase Spipo11G0028200 7 32.7 4.4 down- Ethylene-responsive transcription NO YES regulated factor 4 Spipo8G0035200 7 71.3 9.6 down- Putative nuclease HARBI1-like NO YES regulated Spipo0G0152100 7 15.2 2.1 down- Beta-glucosidase-like glycosyl NO NO regulated hydrolase Spipo3G0031200 7 5.9 0.8 down- Endo-1,4-beta-glucanase NO NO regulated Spipo0G0000400 7 108.8 15.0 down- Short-chain dehydrogenase/ YES YES regulated reductase 2 Spipo9G0039400 7 68.2 9.4 down- Histone H3 NO NO regulated Spipo9G0045200 7 18.8 2.6 down- Carboxyvinyl- NO YES regulated carboxyphosphonate- phosphorylmutase Spipo1G0125300 7 186.7 26.8 down- Lectin NO NO regulated Spipo0G0046100 7 112.4 16.4 down- Histone H3 YES YES regulated Spipo4G0069000 7 23.5 3.5 down- Tyrosine-rich hydroxyproline-rich NO NO regulated glycoprotein Spipo10G0030500 7 7.2 1.1 down- Subtilisin-like serine protease NO NO regulated Spipo8G0045500 7 11.3 1.7 down- WRKY transcription factor, putative NO NO regulated Spipo18G0023200 7 13.3 2.0 down- Naphthoate synthase, putative NO YES regulated Spipo0G0068600 7 168.0 25.6 down- Fanconi anemia group I protein NO NO regulated Spipo32G0009000 7 83.6 12.8 down- G-type lectin S-receptor-like NO YES regulated serine/threonine-protein kinase Spipo4G0061000 6 10.4 1.6 down- GDSL esterase/lipase NO NO regulated Spipo7G0024900 6 2151.8 336.5 down- Auxin-repressed protein NO NO regulated Spipo14G0048100 6 229.3 36.3 down- Aminomethyltransferase NO NO regulated Spipo14G0022500 6 4.7 0.7 down- Disease resistance protein NO NO regulated (CC-NBS-LRR) Spipo4G0004200 6 47.5 7.6 down- Acid phosphatase, putative, expressed NO NO regulated Spipo29G0003800 6 24.4 3.9 down- Heavy metal transport/detoxification NO YES regulated superfamily protein Spipo20G0028500 6 15.7 2.6 down- Sulfate transporter NO NO regulated Spipo9G0012700 6 246.1 40.5 down- Adenylosuccinatesynthetase NO NO regulated Spipo3G0061200 6 2685.1 442.0 down- Chlorophyll a/b-binding protein YES NO regulated Spipo3G0041200 6 65.9 11.0 down- CASP-like protein YES YES regulated Spipo0G0035400 6 33.9 5.7 down- Pectinesterase NO NO regulated Spipo2G0072200 6 38.3 6.4 down- Protein of unknown function, NO NO regulated DUF642 Spipo3G0023000 6 14.9 2.6 down- Cyclin B1 NO YES regulated Spipo3G0026500 6 11.4 2.0 down- Triacylglycerol lipase, putative NO YES regulated Spipo2G0093200 6 44.1 7.6 down- Unknown protein NO NO regulated Spipo13G0007500 6 159.5 27.5 down- Histone H3 YES YES regulated Spipo12G0058200 6 2360.0 407.0 down- Xyloglucanendotransglucosylase/ NO NO regulated hydrolase Spipo5G0067200 6 14.6 2.6 down- Cellulose synthase-like protein NO YES regulated Spipo20G0004100 6 91.6 16.1 down- Nucleoside diphosphate kinase NO NO regulated Spipo0G0015500 6 8.4 1.5 down- Urea active transporter-like protein NO NO regulated Spipo29G0004700 6 10.5 1.9 down- Cyclin B1 NO YES regulated Spipo2G0022100 6 1774.3 315.0 down- Dynein light chain 1 cytoplasmic-like NO YES regulated protein Spipo8G0020200 6 651.1 117.2 down- Thylakoid membrane phosphoprotein NO NO regulated 14 kDa Spipo3G0020200 5 21.1 3.8 down- Alpha-L-arabinofuranosidase YES YES regulated Spipo1G0117200 5 117.9 21.5 down- Laccase 11 NO NO regulated Spipo8G0039700 5 22.8 4.3 down- Glucan endo-1,3-beta-glucosidase, NO YES regulated putative Spipo28G0019000 5 77.9 14.8 down- Histone H4 NO YES regulated Spipo2G0093000 5 50.4 9.6 down- Unknown protein NO YES regulated Spipo28G0012500 5 328.4 64.0 down- Chaperone protein dnaJ YES YES regulated Spipo0G0114100 5 259.2 50.5 down- Protodermal factor 1.3 NO YES regulated Spipo23G0013400 5 476.1 93.1 down- Ribulose-1 5-bisphosphate NO YES regulated carboxylase/oxygenaseactivase Spipo12G0016000 5 10.2 2.0 down- O-fucosyltransferase family protein NO YES regulated Spipo26G0012100 5 894.9 176.9 down- Arabinogalactan protein 20 NO YES regulated Spipo31G0007300 5 3.4 0.7 down- Leucine-rich repeat receptor-like NO NO regulated protein kinase family protein Spipo8G0005400 5 14.9 3.0 down- Glutamate receptor NO NO regulated Spipo1G0080800 5 2505.2 507.5 down- Unknown protein YES NO regulated Spipo3G0012100 5 5.4 1.1 down- Protein kinase-like protein NO NO regulated Spipo20G0028600 5 9.6 2.0 down- Sulfate transporter, putative NO NO regulated Spipo17G0045100 5 86.3 17.8 down- Aquaporin NO YES regulated Spipo2G0059000 5 5.8 1.2 down- Major facilitator superfamily protein NO YES regulated Spipo9G0038700 5 53.8 11.2 down- U-box domain-containing protein NO NO regulated Spipo1G0126300 5 #### 293.1 down- 60S ribosomal protein L10-like NO NO regulated protein Spipo3G0024100 5 24.6 5.3 down- Nicotianamine synthase, putative NO NO regulated Spipo16G0011700 5 25.7 5.5 down- L-lactate dehydrogenase NO NO regulated Spipo6G0071400 5 125.5 27.1 down- Pectinacetylesterase family protein NO YES regulated Spipo22G0026300 5 186.4 40.4 down- Expansin NO YES regulated Spipo2G0039900 5 15.3 3.3 down- Amine oxidase, putative NO YES regulated Spipo2G0092600 5 66.7 14.6 down- Early nodulin-like protein 17 YES YES regulated Spipo0G0183100 5 14.4 3.2 down- Heparanase-like protein 2 NO YES regulated Spipo15G0035800 5 429.5 94.0 down- Glutamine synthetase NO YES regulated Spipo17G0007500 5 101.0 22.2 down- Methyltransferase type 11 NO YES regulated Spipo22G0045000 5 13.9 3.1 down- Mitochondrial carrier family NO YES regulated Spipo13G0011300 4 3034.8 676.8 down- Ferredoxin I NO NO regulated Spipo9G0063000 4 13.1 2.9 down- Beta-galactosidase NO YES regulated Spipo3G0083800 4 297.7 66.7 down- Bifunctional inhibitor/lipid-transfer NO NO regulated protein/seed storage 2S albumin superfamily Spipo29G0021000 4 61.1 14.2 down- PsbP-like protein 2 NO YES regulated Spipo2G0055800 4 17.1 4.0 down- bZIP transcription factor I NO YES regulated Spipo3G0101700 4 12.6 3.0 down- Receptor kinase NO YES regulated Spipo29G0004100 4 288.3 68.2 down- unknown protein NO YES regulated Spipo11G0012500 4 104.1 24.7 down- Antiholin-like protein LrgB NO NO regulated Spipo11G0008200 4 1110.6 266.8 down- Acyl-CoA-binding protein NO YES regulated Spipo9G0043500 4 42.3 10.2 down- Phosphatidylinositol transfer protein YES YES regulated SFH5 Spipo23G0035900 4 68.8 16.6 down- Omega-3 fatty acid desaturase NO YES regulated Spipo27G0016000 4 50.1 12.1 down- Ubiquitin-conjugating enzyme E2 C, NO NO regulated putative Spipo7G0026400 4 32.6 7.9 down- DNA-3-methyladenine glycosylase NO YES regulated Spipo20G0006200 4 7376.0 1797.6 down- Acyl-CoA dehydrogenase NO NO regulated Spipo9G0025800 4 15.7 3.9 down- D-arabinono-1,4-lactone oxidase-like NO YES regulated protein Spipo0G0009000 4 40.9 10.2 down- Fructose-bisphosphatealdolase NO YES regulated Spipo4G0036100 4 49.7 12.3 down- Pectatelyase NO NO regulated Spipo11G0009400 4 38.9 9.7 down- Mitochondrial carrier protein NO NO regulated

TABLE S2 FPKM FPKM Average Average in in FPKM in FPKM in Pathway Gene ID Enzyme frond turion frond turion Lignin Spipo0G0185100 CCR1 32.6 23.5 23 41 Spipo11G0026200 CCR2 3.4 10.2 Spipo6G0037000 CCR3 45.3 35.5 Spipo28G0002300 CCR4 1.0 0.9 Spipo23G0040600 CCR5 6.7 16.7 Spipo11G0026400 CCR6 5.3 10.8 Spipo10G0016700 CCR7 0.3 0.6 Spipo10G0000200 CCR8 26.2 167.8 Spipo8G0071400 CCR9 20.0 24.2 Spipo5G0064600 CCR10 16.7 18.6 Spipo7G0010700 CCR11 10.1 64.8 Spipo0G0172200 CCR12 215.3 306.7 Spipo7G0010800 CCR13 0.4 1.4 Spipo14G0054900 CCR14 2.0 9.9 Spipo12G0004300 CAD1 18.1 32.5 Spipo17G0012300 CAD2 10.8 7.8 Spipo1G0069500 CAD3 2.6 0.7 Spipo2G0124600 CAD4 3.9 2.3 Starch Spipo28G0001400 APS1 264.1 242.5 70 86 Spipo3G0049000 APL1 127.9 18.6 Spipo6G0024200 APL2 23.1 34.2 Spipo18G0038500 APL3 36.0 291.5 Spipo26G0026900 SSI 21.7 28.6 Spipo0G0050800 SSII 3.4 1.6 Spipo14G0048800 SSIII 33.4 24.0 Spipo14G0042000 SSIV 45.7 15.6 Spipo1G0057900 GBSSI 327.8 333.2 Spipo1G0057400 BEI 19.6 10.3 Spipo0G0008100 BEII 40.8 72.0 Spipo12G0062400 ISA1 2.8 14.6 Spipo3G0051400 ISA2 8.1 12.9 Spipo20G0022100 ISA3 25.1 98.0 Lipid Spipo0G0127900 ACCase1 24.0 17.4 28 22 Spipo10G0023400 ACCase2 6.3 4.6 Spipo12G0034900 ACCase3 4.5 2.5 Spipo12G0063600 ACCase4 85.6 44.3 Spipo15G0009000 ACCase5 22.4 21.4 Spipo4G0043600 ACCase6 20.9 11.0 Spipo4G0047600 ACCase7 73.7 56.7 Spipo30G0006700 GPAT1 127.7 52.2 Spipo7G0013300 GPAT2 21.0 20.4 Spipo3G0111400 AGPAT1 1.4 0.3 Spipo4G0068200 AGPAT2 17.6 18.2 Spipo6G0030100 AGPAT3 15.8 13.5 Spipo7G0018900 AGPAT4 4.3 3.3 Spipo7G0051900 AGPAT5 1.6 0.9 Spipo21G0027500 DGAT1 6.0 5.7 Spipo28G0006400 DGAT2 70.9 117.6 Spipo1G0066600 DGAT3 0.9 1.5 Spipo20G0011900 DGAT4 11.2 8.2 Spipo3G0079500 DGAT5 14.6 23.1

The data presented in Table S2 reveal suitable targets for altering carbon partitioning in duckweed. For example, reducing expression of GPAT1 in fronds should increase protein content in duckweed cells. In another approach, introduction of a LEA promoter operably linked to an agent (e.g., an siRNA) to APL1 should increase the lipid content of in the resulting duckweed culture. In yet another approach, one could simultaneously target two or more genes in duckweed which are differentially expressed to alter carbon partitioning in the resulting cells. Methods for introducing transgenes into Duckweed are described in Cantó-Pastor, A., Mollá-Morales, A., Ernst, E., Dahl, W., Zhai, J., Yan, Y., Meyers, B. C., Shanklin, J., Martienssen, R. (2014), Efficient transformation and artificial miRNA gene silencing in Lemna minor. Plant Biology. doi: 10.1111/plb.12215. Sequence information is available on the world wide web at Waksman.rutgers.edu/spirodela/genome.

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims. 

What is claimed is:
 1. A method for altering carbon partitioning from starch to lipids in biomass produced from Duckweed cultures, comprising introducing an agent which modulates expression of at least one gene product identified in Table S1 or Table S2, said agent being effective to reduce starch production and increase lipid production in said culture relative to control untreated cultures.
 2. The method of claim 1, wherein said agent inhibits or increases expression of at least one gene product identified in Table S1 or Table S2, the inhibition or increase resulting in increased lipid production in biomass obtained from said Duckweed culture.
 3. The method of claim 2, wherein said agent inhibits expression of at least one gene selected from the group consisting of AGPS1, AGPL3, GBSSI, ACCase4, GPAT1, APL1, and DGAT2.
 4. The method of claim 1, wherein said agent modulates production of at least one gene in Table S2, thereby increasing lipid biosynthesis and, or protein biosynthesis relative to control untreated cells.
 5. A Duckweed plant produced from the method of claim
 1. 6. The Duckweed plant of claim 5, which is Spirodelapolyrhiza.
 7. A plant part, progeny, seed or cell obtained from the plant of claim
 6. 8. The method of claim 4, wherein said duckweed is cultured under conditions promoting turion formation, and said agent is a transgene comprising a LEA promoter operably linked to an siRNA targeting the APL3 gene, said agent causing a down modulation of APL3 expression, thereby increasing lipid content of said turion.
 9. The method of claim 4, wherein said duckweed is maintained as a frond in culture and said agent is transgene comprising a rubisco promoter operably linked to an siRNA targeting the GPAT1 gene, said agent causing down modulation of GPAT expression, thereby increasing protein content in said frond.
 10. An transgenic duckweed as claimed in claim
 8. 11. A transgenic duckweed as claimed in claim
 9. 12. The method of claim 3, wherein said duckweed comprises at least two agents which modulate expression of GPAT1 and APL1. 